Project Summary Human obesity represents a serious world-wide health problem that is associated with metabolic syndrome and the development of non-alcoholic fatty liver disease (NAFLD) that can progress to non-alcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular carcinoma (HCC). The estimated prevalence of NAFLD in the USA is approximately 25% of the population (1). Estimates for the prevalence of NASH are confounded by the limited availability of reliable non-invasive methods for diagnosis, but approximately 25% of patients with NAFLD exhibit NASH (1). The development of scarring (cirrhosis) and hepatic fibrosis contributes to the development of HCC, the most common liver cancer and the 3rd leading cause of cancer-related death in the USA (2, 3). The high incidence of NAFLD/NASH represents a major health problem because: 1) NASH is anticipated to become the leading indication for liver transplantation (4); and 2) NAFLD/NASH-associated HCC is the primary cause of obesity-related cancer death in the USA (4). Lifestyle interventions, including dietary calorie restriction and exercise, are key aspects of current therapy. However, there is an unmet need for effective pharmacotherapy. Several medications are currently under development, including approaches to reduce steatosis, correct intestinal dysbiosis, promote oxidative stress defense, and suppress fibrosis (5, 6). Recent studies have established that the oxidative stress-responsive protein kinase ASK1 (a member of the MAP3K group) is a promising drug target for the treatment of NASH (5, 6). The small molecule Selonsertib is a potent inhibitor of ASK1 protein kinase activity that causes reduced NASH-related hepatic fibrosis (7, 8), a key determinant of disease progression (4). Successful phase 2 trials of Selonsertib in NASH patients (7, 8) have led to phase 3 trials (STELLAR 3 and STELLAR 4) that are currently in progress (8). The mechanism that accounts for the beneficial effects of blocking ASK1 is unknown, but likely involves a reduction in the activation state of down-stream signaling pathways (e.g. stress-activated MAPK). ASK1 is expressed ubiquitously. Consequently, no information is available concerning the hepatic cell type that mediates the ASK1-promoted hallmarks of NASH, including hepatic fibrosis. It is possible that ASK1 plays a key role in an inflammatory response (e.g. in Kupffer cells and other immune cells) that drives hepatic fibrosis (9). Alternatively, ASK1 may play an important role in steatotic hepatocytes that promotes fibrosis (10). It is also possible that the key role of ASK1 may be in stellate cells that are directly involved in hepatic fibrosis (11). The relevant hepatic cell type that mediates the essential function of ASK1 in NASH has therefore not been defined. Moreover, the mechanism that mediates ASK1 regulation during the development of NASH is not understood. This proposal is designed to identify the mechanism of ASK1 signaling during NASH development. Completion of this study will provide important new information concerning the mechanism of action of drugs that inhibit ASK1. Two Specific Aims are proposed: 1) To identify the hepatic cell type that mediates the effects of ASK1 on hallmarks of NASH; and 2) To identify hepatic mechanisms that regulate ASK1 during NASH development. The overall goal of this research program is to identify molecular mechanisms that account for the function of ASK1 in NASH. Achievement of this goal will increase understanding of NASH development. We anticipate that the successful completion of this research program will lead to the identification of new molecular mechanisms. This knowledge may represent a basis for the design of novel therapeutic strategies for the treatment of NASH, including the rational design of combination therapies.